Recruitment of Armitage and Yb to a transcript triggers its phased processing into primary piRNAs in Drosophila ovaries

Pandey, Radha Raman; Homolka, David; Chen, Kuan-Ming; Sachidanandam, Ravi; Fauvarque, Marie-Odile; Pillai, Ramesh S.

doi:10.1371/journal.pgen.1006956

Cited by 62 publications

(72 citation statements)

References 39 publications

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“…In particular, in clusters 1, 4, and 6 that included transcripts strongly enriched upon CHX treatment and depleted upon PUR treatment, > 90% of RNAs (N = 128/140) code for mitochondrial proteins. While 7 of the remaining 12 transcripts were pseudogenes, of the 5 mRNAs that were in those clusters but not in MitoCarta 2.0, at least 3 are likely to be mitochondrial ( Figure S7E-F) based on other studies (Mou et al, 2009;Pandey et al, 2017;Thul et al, 2017). Similarly, clusters 5+7 that were depleted upon PUR treatment but not enriched by CHX were strongly depleted for mitochondrial genes (<5%, N = 45/990).…”

Section: Distinct Mechanisms For Mrna Localization To the Outer Mitocmentioning

confidence: 73%

Atlas of Subcellular RNA Localization Revealed by APEX-seq

Fazal

Han

Kaewsapsak

et al. 2018

Preprint

112

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We introduce APEX-seq, a method for RNA sequencing based on spatial proximity to the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome, revealing extensive and exquisite patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.(ERM), which we previously attempted but failed to map using APEX2-ERM and APEX-RIP (Kaewsapsak et al., 2017). When we performed a comparison of APEX-seq and APEX-RIP using HEK 239T cells expressing APEX on the ER membrane (facing the cytosol), we found that APEX-seq could clearly enrich secretory mRNAs on the ER membrane over cytosolic mRNAs, whereas APEX-RIP could not ( Figure 1D). Given the very close proximity between ER membrane-associated RNAs and cytosolic RNAs that are immediately adjacent to the ER, these results suggest that APEX-seq has a labeling radius of only a few nanometers. Validation of APEX-seqEncouraged by the results above, we expanded APEX-seq to nine distinct subcellular locations ( Figure 1E). Correct localization of each APEX2 fusion protein (domain structures shown in Figure S2A) in HEK-293T cells was confirmed by imaging with organelle markers ( Figure 1F). We prepared two replicates for each location ( Figure S2B and S2C, correlation coefficient r between replicates ranged from 0.97 to 1) as well as negative controls in which H2O2 was omitted from the labeling reaction. After labeling for 1 minute and cell lysis, we enriched biotinylated RNAs and prepared APEX-seq libraries with polyA+ selection. Transcripts shorter than 100 nt were excluded during purification. We used DESeq2 (Love et al., 2014) for data analysis and used a qvalue (FDR-adjusted p-value) < 0.05 and a log2fold-change > 0.75 to select for significantly enriched transcripts.Consistent with the RT-qPCR results for the mitochondrial matrix shown in Figure 1C, our sequencing-based analysis showed that all 13 mRNAs and 2 rRNAs encoded by mtDNA are strongly enriched by MITO-APEX2 labeling (Figure 2A, S2D and S2E, mean enrichment > 11fold), whereas no mRNAs encoded by the nuclear genome are enriched. Figure 2B shows good correlation (r > 0.9) between technical replicates.To assess APEX-seq in an "open" subcellular region (not enclosed by a membrane), we focused again on the ER membrane (ERM). The ERM is particularly valuable for methodology validation because there are well-established "true positives" (mRNAs encoding secreted proteins that are known to be translated at the ERM) and well-established "false positive...

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Section: Distinct Mechanisms For Mrna Localization To the Outer Mitocmentioning

confidence: 73%

Atlas of Subcellular RNA Localization Revealed by APEX-seq

Fazal

Han

Kaewsapsak

et al. 2018

Preprint

112

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“…Among these is Armitage (Armi), an RNA helicase of the Upf1 family, which localises to nuage and mitochondria in germ cells and predominantly to Ybbodies in follicle cells (Malone et al 2009;Olivieri et al 2010;Saito et al 2010). Armi shows ATP-dependent 5'-3' helicase activity (Pandey et al 2017) and Zuc-mediated piRNA biogenesis, but not the ping-pong cycle, collapses upon its loss. Tethering of Armi to a reporter transcript results in conversion of the RNA into ~25-nt piRNAs (Pandey et al 2017;Rogers et al 2017).…”

Section: Introductionmentioning

confidence: 99%

“…Armi shows ATP-dependent 5'-3' helicase activity (Pandey et al 2017) and Zuc-mediated piRNA biogenesis, but not the ping-pong cycle, collapses upon its loss. Tethering of Armi to a reporter transcript results in conversion of the RNA into ~25-nt piRNAs (Pandey et al 2017;Rogers et al 2017). The mouse homolog of Armi, MOV10L1, also binds to piRNA precursors and initiates the production of piRNAs (Vourekas et al 2015).…”

Section: Introductionmentioning

confidence: 99%

Daedalus and Gasz recruit Armitage to mitochondria, bringing piRNA precursors to the biogenesis machinery

Munafò

Manelli

Falconio

et al. 2019

Preprint

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The piRNA pathway is a small RNA-based immune system that silences mobile genetic elements in animal germlines. piRNA biogenesis requires a specialised machinery that converts long single-stranded precursors into small RNAs of ~25-nucleotides in length. This process involves factors that operate in two different subcellular compartments: the nuage/Yb-body and mitochondria. How these two sites communicate to achieve accurate substrate selection and efficient processing remains unclear. Here, we investigate a previously uncharacterized piRNA biogenesis factor, Daedalus (Daed), that is located on the outer mitochondrial membrane. Daed is essential for Zucchini-mediated piRNA production and for the correct localisation of the indispensable piRNA biogenesis factor, Armitage (Armi).We find that Gasz and Daed interact with each other and likely provide a mitochondrial "anchoring platform" to ensure that Armi is held in place, proximal to Zucchini, during piRNA processing. Our data suggest that Armi initially identifies piRNA precursors in nuage/Ybbodies in a manner that depends upon Piwi and then moves to mitochondria to present precursors to the mitochondrial biogenesis machinery. These results represent a significant step in understanding a critical aspect of transposon silencing, namely how RNAs are chosen to instruct the piRNA machinery in the nature of its silencing targets.

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“…Phased piRNA production also requires Armitage (Armi), a member of the Upf1 family of ATP-dependent 5′-to-3′ helicase proteins (Cook et al, 2004;Haase et al, 2010;Olivieri et al, 2010;Saito et al, 2010). Strikingly, artificially tethering Armi to a transcript triggers its conversion into piRNAs (Pandey et al, 2017;Rogers et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

The RNA-binding ATPase, Armitage, Couples piRNA Amplification in Nuage to Phased piRNA Production on Mitochondria

Wang

Tipping

et al. 2018

Preprint

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SUMMARYPlWI-interacting RNAs (piRNAs) silence transposons in Drosophila ovaries, ensuring female fertility. Two coupled pathways generate germline piRNAs: the ping-pong cycle, in which the PIWI proteins Aubergine and Ago3 increase the abundance of pre-existing piRNAs, and the phased piRNA pathway, which generates strings of tail-to-head piRNAs, one after another. Proteins acting in the ping-pong cycle localize to nuage, whereas phased piRNA production requires Zucchini, an endonuclease on the mitochondrial surface. Here, we report that Armitage (Armi), an RNA-binding ATPase localized to both nuage and mitochondria, links the ping-pong cycle to the phased piRNA pathway. Mutations that block phased piRNA production deplete Armi from nuage. Armi ATPase mutants cannot support phased piRNA production and inappropriately bind mRNA instead of piRNA precursors. We propose that Armi shuttles between nuage and mitochondria, feeding precursor piRNAs generated by Ago3 cleavage into the Zucchini-dependent production of Aubergine- and Piwi-bound piRNAs on the mitochondrial surface.

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Recruitment of Armitage and Yb to a transcript triggers its phased processing into primary piRNAs in Drosophila ovaries

Cited by 62 publications

References 39 publications

Atlas of Subcellular RNA Localization Revealed by APEX-seq

Atlas of Subcellular RNA Localization Revealed by APEX-seq

Daedalus and Gasz recruit Armitage to mitochondria, bringing piRNA precursors to the biogenesis machinery

The RNA-binding ATPase, Armitage, Couples piRNA Amplification in Nuage to Phased piRNA Production on Mitochondria

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